Synthetic HPV16 virus-like particles

ABSTRACT

The invention is a series of synthetic virus-like particles useful in the characterization of human papillomavirus infection and assays employing the synthetic virus-like particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the provisional U.S. applicationSer. No. 06/026,763 filed Oct. 4, 1996.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

Not applicable

REFERENCE TO MICROFICHE APPENDIX

Not applicable

FIELD OF THE INVENTION

The present invention is a series of synthetic virus-like particles(VLP) useful in the characterization of human papillomavirus infectionand assays employing the synthetic virus-like particles.

BACKGROUND OF THE INVENTION

Papillomavirus infections occur in a variety of animals, includinghumans, sheep, dogs, cats, rabbits, monkeys, snakes and cows.Papillomaviruses infect epithelial cells, generally inducing benignepithelial or fibroepithelial tumors at the site of infection.Papillomaviruses are species specific infective agents; a humanpapillomavirus cannot infect a nonhuman animal.

Papiromaviruses may be classified into distinct groups based on the hostthat they infect. Human papillomaviruses (HPV) are further classifiedinto more than 60 types based on DNA sequence homology (for a review,see Papillomaviruses and Human Cancer, H. Pfister (ed.), CRC Press,Inc., 1990). Papillomavirus types appear to be type-specific immunogensin that a neutralizing immunity to infection to one type ofpapillomavirus does not confer immunity against another type ofpapillomavirus.

In humans, different HPV types cause distinct diseases. HPV types 1, 2,3, 4, 7, 10 and 26-29 cause benign warts in both normal andimmunocompromised individuals. HPV types 5, 8, 9, 12, 14, 15, 17, 19-25,36 and 46-50 cause flat lesions in immunocompromised individuals. HPVtypes 6, 11, 34, 39, 41-44 and 51-55 cause nonmalignant condylomata ofthe genital or respiratory mucosa. HPV types 16 and 18 cause epithelialdysplasia of the genital mucosa and are associated with the majority ofin situ and invasive carcinomas of the cervix, vagina, vulva and analcanal. HPV6 and HPV11 are the causative agents for more than 90% of allcondyloma (genital warts) and laryngeal papillomas.

Immunological studies in animals have shown that the production ofneutralizing antibodies to papillomavirus antigens prevents infectionwith the homologous virus. The development of effective papillomavirusvaccines has been slowed by difficulties associated with the cultivationof papillomaviruses in vitro. The development of an effective HPVvaccine has been particularly slowed by the absence of a suitable animalmodel. Neutralization of papillomavirus by antibodies appears to betype-specific and dependent upon conformational epitopes on the surfaceof the virus.

Papillomaviruses are small (50-60 nm), nonenveloped, icosahedral DNAviruses that encode for up to eight early and two late genes. The openreading frames (ORFs) of the virus genomes are designated E1 to E7 andL1 and L2, where "E" denotes early and "L" denotes late. L1 and L2 codefor virus capsid proteins. The early (E) genes are associated withfunctions such as viral replication and cellular transformation.

The L1 protein is the major capsid protein and has a molecular weight of55-60 kDa. L2 protein is a minor capsid protein which has a predictedmolecular weight of 55-60 kDa and an apparent molecular weight of 75-100kDa as determined by polyacrylamide gel electrophoresis. Immunologicdata suggest that most of the L2 protein is internal to the L1 protein.The L2 proteins are highly conserved among different papillomaviruses,especially the 10 basic amino acids at the C-terminus. The L1 ORF ishighly conserved among different papillomaviruses.

The L1 and L2 genes have been used to generate vaccines for theprevention and treatment of papiromaviruses infections in animals. Zhouet al., (1991; 1992) cloned HPV type 16 L1 and L2 genes into a vacciniavirus vector and infected CV-1 mammalian cells with the recombinantvector to produce virus-like particles (VLP).

Recombinant baculoviruses expressing HPV6 L1, HPV11 L1, HPV16 L1, HPV18L1, HPV31 L1 or HPV16 L2 ORFs have been used to infect insect Sf9 cellsand produce L1 and L2 proteins. Western blot analyses showed that thebaculovirus-derived L1 and L2 proteins reacted with antibody to HPV16.The baculovirus derived L1 forms VLPs.

Carter et al., (1991) demonstrated the production of HPV 16 L1 and HPV16L2 proteins by recombinant strains of Saccharomvces cerevisiae. Carteret al. also demonstrated the production of HPV6b L1 and L2 proteins. TheHPV6b L1 protein was not full-length L1 protein. The recombinantproteins were produced as intracellular as well as secreted products.The recombinant L1 and L2 proteins were of molecular weights similar tothe native proteins. When the proteins were expressed intracellularly,the majority of the protein was found to be insoluble when the cellswere lysed in the absence of denaturing reagents. Although thisinsolubility may facilitate purification of the protein, it may hamperanalysis of the native epitopes of the protein.

Recombinant proteins secreted from yeast were shown to containyeast-derived carbohydrates. The presence of these N-linkedoligosaccharides may mask native epitopes. In addition, the secretedrecombinant proteins may contain other modifications, such as retentionof the secretory leader sequence.

The present invention is directed to the production of recombinantpapillomavirus proteins having the immunity-conferring properties of thenative papillomavirus proteins as well as methods for their productionand use. The present invention is a series of synthetic virus-likeparticles useful in the characterization of human papillomavirusinfection and assays employing the synthetic virus-like particles.

The invention involves the delineation of residues specific to HPV11 L1which are required for binding neutralizing antibodies, and a modifiedHPV16 L1 gene with HPV11-like substitutions such that VLPs produced fromthe modified HPV16 L1 gene also bind HPV11 neutralizing monoclonalantibodies.

We previously demonstrated that HPV11 L1 residues Gly¹³¹ -Tyr¹³² wereresponsible for the HPV11 specificity of binding of several HPV11neutralizing monoclonal antibodies. Because the binding of theseantibodies is conformationally dependent, it remained unanswered as towhether the epitope is continuous and comprised of residues located nextto each other with conformation requiring VLP assembly, or discontinuousand comprised of residues well separated on the L1 linear sequence butwhich come into close proximity upon proper folding and assembly ofparticles. We scanned residues over a 20 residue stretch centered atGly¹³¹ -Tyr¹³², and identified five residues where substitution resultedin significant loss of binding of the neutralizing monoclonalantibodies, without affect on other HPV11 specific, VLP-dependentantibodies. This demonstrates that the epitope is continuous. This wasconfirmed by demonstrating that HPV11 substitutions at these positionsinto the HPV16 L1 sequence forms the basis of transfer of binding ofthese monoclonal antibodies to modified HPV16 VLPs.

The panel of neutralizing monoclonal antibodies for HPV11 was obtainedfrom Neil Christensen (Pennsylvania State University, Hershey, Pa). Themonoclonal antibodies in the panel are HPV11 specific and VLP-dependent.The antibodies may be distinguished from each other in terms of whichamino acid residues affect binding of the individual antibodies,although there are overlapping positions for all the monoclonalantibodies. Additional antibodies used in these studies were alsoobtained from Dr. Neil Christensen.

These residues collectively define the epitope for antibodies known toneutralize HPV11 . We also demonstrate that substitution of theseresidues into equivalent positions of the HPV16 L1 sequence form thebasis of transferring binding of these antibodies to modified HPV16VLPs. The modified HPV16 VLPs may be used to develop HPV11 specificserological assays. Because of the high identity between HPV6 and HPV11L1 sequences, present serological assays cannot distinguish responsesbetween these two types very well. Modified HPV16 VLPs with a singleHPV11 specific epitope and no cross-reactivity to HPV6 VLPs should beable to identify HPV11 immune responses upon infectivity orimmunization.

This problem has not been solved in the past and, to our knowledge, isthe first demonstration that a conformationlly dependent epitope iscontinuous.

There were two difficulties to overcome. First, the epitope isconformational, and conventional means of epitope mapping, binding topeptide fragments, could not be utilized. It was necessary to expressany test L1 protein in a way that facilitated formation of virus-likeparticles which minic the virus structure. Second, the large number ofL1 clones required for the mapping necessitated the generation of afacile means to express the test viral coat proteins.

Without isolation of a type-specific epitope, it would be difficult todistinguish HPV6 and HPV11 immune responses.

One use of the derivatized HPV16 VLP is as a reagent in a serologicalassay. Because most epitopes are shared between HPV6 and HPV11 VLPs,polyclonal sera to one competes with the binding of a type-specificmonoclonal antibody to the other due to steric hindrance from thebinding of antibodies to neighboring sites. There are very fewcross-reactive epitopes between HPV16 and either HPV11 or HPV6.Therefore, presentation of an HPV11 specific epitope on an HPV16 VLPshould eliminate the problem of steric competition from neighboringepitopes. Only the presence of antibody in a polyclonal response to thespecifically transferred epitope should compete with monoclonal antibodybinding.

SUMMARY OF THE INVENTION

This invention includes a series of synthetic virus-like particles (VLP)useful in the characterization of human papillomavirus types 11 and 16infection and assays employing the synthetic particles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequences of the HPV11 and HPV16 L1 proteinin the region mutagenized (residues 121 through 147), and also shows thespecific substitutions made in this study. These sequences are availablein the EMBL Gene Bank. The lower line shows the differences with the HPVL1 sequence over the homologous stretch. Note that the alignment shiftsthe HPV16 sequence three residues with respect to HPV11. A G¹³¹ :Y¹³²double substitution into the HPV6 L1 sequence was previouslydemonstrated to be necessary and sufficient to transfer binding of HPV11neutralizing MAbs to HPV6 VLPs. Substitutions into the HPV11 sequencewere individually made and analyzed for MAb binding at all underlinedpositions. Where the two sequences differ, HPV16-like substitutions weremade. Otherwise alanine was substituted. Substitutions which resulted inloss of binding of one or more HPV11 neutralizing MAb are indicated bybold-face.

FIG. 2 shows the amino acid sequences or the L1 protein of HPV16 andseveral substituted clones (HPV16:5, HPV16:8 and HPV16:10). HPV16:5contains five HPV11-like substitutions in positions demonstrated ascritical to the binding of one or more HPV11 neutralizing MAbs. HPV16:8contains three additional substitutions in residues non-critical for MAbbinding. These latter substitutions are all alanine to glycine oralanine to proline substitutions, residues known to strongly perturblocal structure. HPV16:10 contains a further two 11-like substitutionsto generate a stretch of residues which completely matches the HPV11 L1sequence over the region. FIGS. 3 and 4 show the results of MAb bindingto VLPs produced from these clones.--.

FIG. 3 shows that amino acid substitutions at critical positions are notsufficient to transfer HPV11 monoclonal antibody (MAb) binding to HPV16VLPs. VLPs for HPV11, HPV16 and HPV16:5 (HPV16 with five 11-likesubstitutions) were expressed in Sf9 cells and assayed for binding byHPV11 or HPV16 specific MAbs. The five substitutions into HPV16 L1 arewithout affect on the binding of two HPV16 specific MAbs, but also ofnot result in transfer of HPV11 MAb binding. MAbs were used as 10⁻⁵dilution of ascites stock. Binding to HPV11 extract by MAb CRPV.5A, aCRPV-specific antibody which does not cross-react with humanpapillomavirus types, is shown as a dashed line.

FIG. 4 shows that eight amino acid substitutions into the HPV16 L1sequence confers HPV11 monoclonal antibody binding and demonstrate acontinuous, conformational epitope. VLPs for HPV16, HPV16:8, andHPV16:10 were expressed in Sf9 cells and assayed for binding by HPV11 orHPV16 specific MAbs. HPV16:8 contains five critical substitutions, andthree additional A to G or A to P substitutions in positionsindividually shown not to be critical for binding (see FIG. 2). HPV16:8VLPs, but not HPV16 VLPs, bind HPV11 specific MAbs H11.B2, H11.F1 andH11.G5. Note that binding of MAbs H11.A3 and H11.H3, both of which wereshown not to be affected by substitutions within this stretch, is notobserved. MAbs 11.B2, H11.F1, and H11.G5, whose binding is transferredto HPV16:8 VLPs, bind only assembled virus or VLPs. These three MAbswere previously demonstrated to neutralize infectious HPV11 virus in themouse xenograph assay. HPV16:10 VLPs, which contain two additional HPV11substitutions to make the entire stretch match the HPV11 sequence, donot show any additional level of HPV11 MAb binding. MAbs were used as10³¹ 5 dilution of ascites stock. Bonding to HPV11 extract by MAbCRPV.5A, a CRPV-specific antibody which does not cross-react with humanpapillomavirus types, is shown as a dashed line.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a series of synthetic virus-like particles(VLP) useful in the characterization of human papillomavirus 11infection and assays employing the synthetic virus-like particles, whichmay be used to monitor serological responses to HPV11 infection andimmunization.

Papillomavirus infections occur in a variety of animals, includinghumans, sheep, dogs, cats, rabbits, monkeys, snakes and cows.Papillomaviruses infect epithelial cells, generally inducing benignepithelial or fibroepithelial tumors at the site of infection.

Papillomaviruses may be classified into distinct groups based on thehost that they infect. Human papillomaviruses (HPV) are furtherclassified into more than 60 types based on DNA sequence homology (for areview, see Papillomaviruses and Human Cancer, H. Pfister (ed.), CRCPress, Inc., 1990). Papillomavirus types appear to be type-specificimmunogens in that a neutralizing immunity to infection to one type ofpapillomavirus does not confer immunity against another type ofpapillomavirus.

In humans, different HPV types cause distinct diseases. HPV types 1, 2,3, 4, 7, 10 and 26-29 cause benign warts in both normal andimmunocompromised individuals. HPV types 5, 8, 9, 12, 14, 15, 17, 19-25,36 and 46-50 cause flat lesions in immunocompromised individuals. HPVtypes 6, 11, 34, 39, 41-44 and 51-55 cause nonmalignant condylomata ofthe genital and respiratory mucosa. HPV types 16 and 18 cause epithelialdysplasia of the genital tract and are associated with the majority ofin situ and invasive carcinomas of the cervix, vagina, vulva and analcanal. HPV6 and HPV11 cause the majority of genital warts and laryngealpapillomas.

Immunological studies in animals have shown that the production ofneutralizing antibodies to papillomavirus capsid proteins preventsinfection with the homologous virus. The development of effectivepapillomavirus vaccines has been slowed by difficulties associated withthe cultivation of papillomaviruses in vitro. The development of aneffective HPV vaccine has been particularly slowed by the absence of asuitable animal model. Neutralization of papillomavirus by antibodiesappears to be type-specific and dependent upon conformational epitopeson the surface of the virus.

Papillomaviruses are small (50-60 nm), nonenveloped, icosahedral DNAviruses that encode for up to eight early and two late genes. The openreading frames (ORFs) of the virus genomes are designated E1 to E7 andL1 and L2, where "E" denotes early and "L" denotes late. L1 and L2 codefor virus capsid proteins. The early (E) genes are associated withfunctions such as viral replication and transformation.

The L1 protein is the major capsid protein and has a molecular weight of55-60 kDa. L2 protein is a minor capsid protein which has a predictedmolecular weight of 55-60 kDa and an apparent molecular weight of 75-100kDa as determined by polyacrylamide gel electrophoresis.

The production of HPV16 L1, HPV16 L2, and HPV type 6 L1 proteins byrecombinant strains of Saccharomyces cerevisiae has been reported. Itwould be useful to develop methods of producing large quantities ofpapillomavirus proteins of any species and type by cultivation ofrecombinant yeasts. It would also be useful to produce large quantitiesof papillomavirus proteins having the immunity-conferring properties ofthe native proteins, such as the conformation of the native protein. Toachieve this latter goal it would be necessary to analyze the effect ofnumerous mutations in the L1 gene on the binding of antibodies of knownproperties (VLP dependent, cross-reactive, etc.)

The empirical scanning of natural or engineered peptide sequences forfunctional residues is inherently dependent upon expression of largenumbers of sequence variants to assay their relative functional potency.The level of protein expression obtained can be particularly critical inthe case of self-assembling viral structural proteins, because theefficiency of self-assembly frequently is concentration dependent. Theinsect baculovirus expression vector system has been widely used tostudy viral self-assembly, but it generally requires prior isolation andexpansion of a plaque-purified recombinant viral stock to generateuseful quantities of self-assembled particles. In examining a number ofpossibilities for expression of analytical levels of the L1 coat proteinof Cottontail Rabbit and Human Type 11 Papillomaviruses, we found thateven brief transient cotransfection of insect cells with baculovirustransfer vectors and viral DNA yielded assembled particles which wereimmunologically indistinguishable from particles previously obtainedfrom plaque purified stocks (Benincasa et. al. 1996. Rapid, high-leveltransient expression of papillomavirus-like particles in insect cells.BioTechniques 20,890-895). Within six days of plasmid/viral DNAcotransfection of Sf9 cells, at least 1-2 μg of assembled L1particles/100 mm plate could be demonstrated. This level of expressionis more than sufficient to assay functionality, and has severaladvantages over comparable mammalian cell transient expression systems.

To define neutralizing epitopes in HPV infections, we need to identifythe amino acid residues that confer antigenic type-specificity on humanpapillomavirus subtypes (Christensen, N. D., et. al. 1990, Monoclonalantibody-mediated neutralization of infectious human papillomavirus type11). Many of the type-specific epitopes are conformationally-dependentand are detectable only upon VLP assembly. The L1 structural coatprotein of several animal and human papillomaviruses has beendemonstrated to efficiently self-assemble when expressed in insect cellsvia recombinant baculovirus strains (Christensen, N. D., et al., 1994,Assembled baculovirus-expressed human papillomavirus type 11 L1 capsidprotein virus-like particles are recognized by neutralizing monoclonalantibodies and induce high titres of neutralizing antibodies. J. Gen.Virol. 75, 2271-2276). The time and labor involved in the generation ofrecombinant phage precludes the use of this method to screen a largenumber of VLP variants produced through site-directed mutagenesis.However, we previously observed that when expressed in the baculovirussystem, a recombinant protein is detectable as a secreted product inμg/ml quantities within 5-7 days of the initial transfection of insectcells with plasmid and viral DNAs. Based upon this observation, weexamined whether sufficient quantities of papillomavirus L1 proteinwould accumulate to allow self-assembly into VLPs upon transientexpression, particularly if a more efficient baculovirus transfectionsystem such as the Baculogold™ (Pharmingen, San Diego, Calif.) systemwere utilized. Employing a rapid 6-day transient transfection protocol,the L1 coat protein of numerous papillomavirus types, properly assembledinto VLPs, was produced. Extracts prepared from transiently transfectedcells with CRPV or HPV11 L1 gene constructs contained immunogenicmaterial recognized by type-specific and VLP dependent monoclonalantibodies generated against either CRPV or HPV11 VLPs. The transientlyexpressed material was not cross-reactive with other type-specificantibodies, and recognition was sensitive to alkaline denaturation,further demonstrating fidelity in VLP formation.

We previously identified HPV11 L1 residues Gly¹³¹ -Tyr¹³² as responsiblefor the type-specific binding of several HPV11 neutralizing monoclonalantibodies (Ludmerer et. al. 1996. Two amino acid residues confer typespecificity to a neutralizing, conformationally dependent epitope onhuman papillomavirus type 11. J. Virol. 70, 4791-4794). To map the HPV11neutralizing epitope, we individually mutated HPV11 at the residueswhere the sequence diverges from the HPV16 sequence over a 20 residuestretch centered at Gly¹³¹ -Tyr¹³². The positions were mutated to matchthe HPV16 sequence. Using the Sf9 transient expression system describedabove, these mutant HPV11 L1 genes were expressed and analyzed forbinding by HPV11specific monoclonal antibodies.

The following examples are provided to further define the inventionwithout, however, limiting the invention to the particulars of theseexamples.

EXAMPLE 1

Generation of test expression constructs.

The HPV11 L1 structural gene was cloned from clinical isolates using PCRwith primers designed from the published L1 sequence. The L1 gene wassubsequently subcloned both into BlueScript (Pharmacia) for mutagenesis,and pVL1393 (Stratagene) for expression in Sf9 cells.

Mutations were introduced into the L1 gene using Amersham Sculptor invitro mutagenesis kit. The appearance of the desired mutation wasconfirmed by sequencing, and the mutated gene subdoned into pVL1393 forexpression in Sf9 cells.

The HPV16 L1 structural gene was subdoned both into BluScript(Pharmacia) for mutagenesis, and pVL1393 (Stratagene) for expression inSf9 cells. Mutations were generated using the Amersham Sculptor in vitromutagenesis kit, verified by sequencing, and subcloned into pVL1393 forexpression in Sf9 cells.

EXAMPLE 2

Transient Expression of L1 VLPs in SF9 cells.

SF9 cells were transfected using BaculoGold Transfection kit(Pharmingen). Transfections were done essentially according to themanufacturer's instructions with the following modifications. 8·10⁸ Sf9cells were transfected in a 100 mM dish, with 4 μg of BaculoGold DNA and6 ug of test DNA. Cells were harvested after 6 days and assayed for VLPproduction.

EXAMPLE 3

Preparation of SF9 extracts and ELISA assays.

Cells were harvested six days after transfection, by scraping followedby low speed centrifugation. Cells were resuspended in 300 ml ofbreaking buffer (1 M NaCl, 0.2 M Tris pH 7.6) and homogenized for 30" onice using a Polytron PT 1200 B with a PT-DA 1205/2-A probe (Brinkman) ina Falcon 1259 tube. Samples were spun at 2500 rpm for 3 minutes topellet debris. Tubes were washed with an additional 150 ml of breakingbuffer, supernatants collected in a 1.5 ml microfuge tube, and respunfor 5 minutes in an Eppendorf microfuge (Brinkman). Supernatants werecollected and stored at 4° C. until use. ELISA assays typically wereperformed the same day.

5 ml of extract was diluted into 50 ml of 1% BSA in PBS (phosphatebuffered saline; 20 mM NaPO₄, pH 7.0, 150 mM NaCl) and plated onto apolystyrene plate. The plate was incubated overnight at 4° C. Extractswere removed and the plate blocked with 5% powdered milk in PBS. Allsubsequent wash steps were performed with 1% BSA in PBS. The plate wasincubated at room temperature with primary antibody for 1 hour. Primaryantibodies, monoclonal antibodies generated against HPV11 VLPs, wereobtained as ascites stock from Dr. Neil Christensen (Pennsylvania StateUniversity). They were diluted 10⁵ in 1% BSA PBS before use. Afterwashing, plates were incubated for 1 hour with secondary antibody. Thesecondary antibody, peroxidase labeled Goat anti-Mouse IgG (g), waspurchased from Kirkegaard & Perry Laboratories, Inc. and used at 10³dilution in 1% BSA in PBS. After a final washing, an alkalinephosphatase assay was performed and absorbance read at 405 nm.

EXAMPLE 4

HPV11 scan

To map the residues critical for an HPV11 specific neutralizing epitope,we take advantage of two conditions. First of all, we used a panel ofmonoclonal antibodies which are specific for HPV11 L1 and recognize L1only when assembled into a VLP. Among these five antibodies, 4 have beendemonstrated to neutralize HPV11 in the Kreider Xenograft system(Kreider et al., 1987, J. Virol. 61:590-593)

We previously demonstrated that the type-specificity to binding of threeof the neutralizing MAbs is due to Gly¹³¹ -Tyr¹³² of the HPV11 L1sequence, and that the fourth neutralizing monoclonal antibody binds adifferent site. Because the epitope is conformational, a more completedescription of the epitope is desired. In particular, it was not knownif the epitope is continuous, with contact residues in close proximityin the linear sequence and conformation requiring proper L1 folding andassembly, or discontinuous, with the contact residues at diversepositions of the linear sequence with positional proximity emerging onlyafter assembly.

We reasoned that if the epitope is continuous, then several residuescritical for binding should be located within a short distance of theGly¹³¹ -Tyr¹³² pair. Because a typical linear epitope spans 10-12residues, we scanned residues within 12 positions of Gly¹³¹ -Tyr¹³² foran approximately 25-residue stretch. Because binding is specific forHPV11 VLPs, we focused on those residues where the sequence divergesbetween HPV11 and HPV16. Beyond this 25 residue region the homologybetween the two sequences increase dramatically. Substitutions intoHPV11L1 were selected from the HPV16 sequence to minimize thepossibility that the substitution would result in a more generalperturbation on VLP structure.

To determine the affect on binding of any particular residue, both HPV11and the corresponding HPV11 derivative were expressed in the transientexpression system. An ELISA was performed using the panel of HPV11specific monoclonal antibodies, and results between the two compared. L1production was normalized with monoclonal antibody H6.C6. H6.C6 antibodyis cross-reactive with HPV11, and its epitope is linear and recognizedindependently of VLP formation. Thus it measures L1 production.

Results are put through a double normalization. First, the ratio ofabsorbance of the test antibody to H6.C6 is calculated for the testposition. The same ratio is determined for HPV11 and divided into theratio for the test position. Thus a double ratio near 1 means that thereis no detectable difference in antibody binding to the test clonerelative to HPV11. A double ratio less than one means that the testantibody binds more poorly to the test clone than wild-type. In theory,a ratio greater than 1 means that the antibody binds better to the testclone than to HPV11. In practice this was not observed. A ratio in therange of 0.1 to 0.2 is essentially background, meaning we cannot detectbinding of the antibody to the mutant VLP.

The positions in HPV11 L1 between residue 120 and 145 which differ fromHPV16 L1 were individually substituted with the HPV16 residue. Cloneswere expressed in SF9 cells through a Baculovirus expressingrecombinant, and affect of binding by the panel of HPV11 specificmonoclonal antibodies determined (Table 1). Only substitutions whichresulted in binding impairment for one or more antibodies are includedin the table. Note that the binding of H11.A3.2 and H11.H3 is notimpaired upon any substitution. Both are HPV11 specific, VLP dependentMAbs demonstrated to bind different regions of VLPs than H11.B2, H11.F1,and H11.G5. Binding by H11.A3.2 and H11.H3 verifies the assembly ofVLPs, and demonstrates that the effect of substitution is specific forantibodies binding at this region.

                  TABLE 1    ______________________________________    Position          H11.A3.2   H11.B2  H11.F1  H11.G5                                           H11.H3    ______________________________________    Y123L 0.65       0.48    0.17    0.22  1.34    G130S 0.88       0.44    0.08    0.11  1.3    G131A 0.99       0.11    0.08    0.10  1.03    Q138V 0.93       0.45    0.62    1.05  0.83    V142E 0.93       nd      0.40    0.57  1.27    ______________________________________

EXAMPLE 5

Transfer of the HPV11 Neutralizing Epitope to HPV16

Based upon the studies in Example 4, we mutated the HPV16 L1 gene atamino acid residues 126, 133, 134,141, and 145 to match the HPV11 L1sequence. We designate this clone as HPV16:5 To our surprise, we saw nobinding of HPV11 monoclonal antibodies to VLPs produced from this clone.We reasoned that we had provided the important contact positions, butwere not presenting this highly conformationally dependent epitopecorrectly. We noted that the HPV11 L1 sequence contains the residuesG¹³³, G¹³⁴, and P¹³⁶, where the HPV16 L1 sequence has alanine in thethree equivalent positions 136, 137, and 139. Glycine and prolineresidues can introduce structural perturbations. Although individualsubstitutions into the HPV11 L1 sequence at these positions were withouteffect, we reasoned that three such changes right at the region of theepitope may collectively affect epitope presentation. Using cloneHPV16:5 as a template, we generated a clone that had the furthersubstitutions A136G, A137G, and A139P to create clone HPV16:8. Thisclone produced VLPs which bind HPV11 antibodies H11.B2, H11.F1, andH11.G5. Binding of two separate HPV16 VLP dependent monoclonalantibodies is not affected. Using HPV16:8 as a template, we generated athird clone which has T129V and A132S substitutions to generateHPV16:10. This clone is identical to HPV11 L1 over the region evaluated,but these latter two positions were shown to be nonessential forbinding. Consistent with this, clone HPV16:10 shows no furtherimprovement in MAb binding relative to HPV16:8. Binding is normalized toL1 production using monoclonal antibody H16.D9, which binds an internallinear epitope that is presented only upon denaturation of the sample.The table is divided into two sets because the pairs of samples weretransfected and assayed separately. Binding of HPV11 neutralizingmonoclonal antibodies to modified HPV16 VLPs

    __________________________________________________________________________    Clone         H11.A3.2              H11.B2                  H11.F1                       H11.G5                           H11.H3                               H16.U4                                    H16.V5    __________________________________________________________________________    HPV16         0.07 0.07                  0.07 0.07                           0.13                               1.16 1.7    HPV16:8         0.10 1.51                  0.50 1.27                           0.16                               1.60 2.17    HPV16:10         0.10 1.26                  0.26 1.20                           0.17                               1.70 2.40    HPV16         0.12 0.12                  0.12 0.12                           0.12                               1.0  1.6    HPV16:5         0.11 0.11                  0.12 0.11                           0.11                               0.47 0.85    __________________________________________________________________________

Clone HPV16:8 also demonstrates the mapping of the HPV11 neutralizingepitope, and the potential to use this information to transfer it to adistal surface. In principle, this mapping data could be the basis oftransfer to an even more distal surface, such as CRPV VLPs. Such areagent could be used in the same way as we described for HPV16:8 VLPsin a serological assay, with the added advantage that it could be usedto screen the immunological response of individuals immunized withmultiple VLP types which include HPV6, HPV11 and HPV16 VLPs.

EXAMPLE 6

Monitoring Serological Responses to HPV11 Infection or Immunization

HPV16 modified VLPs are used to determine the presence of an immuneresponse to HPV11 following viral infection or immunization with HPV11VLPs. HPV16 modified VLPs which present the HPV11 neutralizing epitopewill be coated onto the well of a microtitre plate in native form.Following blocking, an HPV11 monoclonal antibody which binds thisepitope, H11.B2, H11.F1, or H11.G5, will be incubated in ELISA formatwith increasing amounts of HPV11 polyclonal sera, HPV6 polyclonal sera,and test polyclonal sera. Binding of the HPV11 monoclonal antibody willbe visualized using a rabbit anti-mouse IgG secondary antibody.Alternatively, it can be labeled with I¹²⁵, or coupled directly to horseradish peroxidase or alkaline phosphatase, or another standard ELISAvisualization protocol. Increasing amounts of polyclonal HPV11 sera willcompete with binding until the signal eventually is reduced tobackground level. Polyclonal HPV6 sera will not compete, or thecompetition will be significantly reduced from that observed with HPV11polyclonal sera. Competition with the test sera at levels comparable toHPV11 polyclonal sera will demonstrate an immune response to HPV11. Lackor significant reduction of competition will demonstrate lack of or aweak immune response to HPV11.

EXAMPLE 7

Transient expression of VLPs in Sf9 cells

The HPV11 L1 structural gene was cloned from clinical isolates using thePolymerase Chain Reaction (PCR) with primers designed from the publishedL1 sequence (8,17). The CRPV L1 structural gene was cloned by PCR fromviral genomic DNA. The L1 genes were subcloned into pVL1393 (Stratagene)for expression in Sf9 cells.

Sf9 cells were cotransfected using the BaculoGold Transfection kit(Pharmingen, San Diego, Calif.). Transfections were done according tothe manufacturer's instructions with the following modification: 8.10⁶Sf9 cells were transfected in a 100 mm dish, with 4 μg of BaculoGoldviral DNA and 6 μg of test plasmid DNA. Cells were harvested after 6days, except where otherwise specified, and assayed for VLP productionby Western Blot or ELISA assay (below).

EXAMPLE 8

Preparation of Sf9 extracts and ELISA assays.

Cells were harvested six days after transfection. Plates were scraped toresuspend cells, and the cells were collected by low speedcentrifugation. Cells were resuspended in 300 μl of breaking buffer (1 MNaCl, 0.2 M Tris pH 7.6) and homogenized for 30 seconds on ice using aPolytron PT 1200 B with a PT-DA 1205/2-A probe (Brinkman) in a Falcon2059 tube. Samples were spun at 2500 rpm in a GPR centrifuge (BeckmanInstruments, Inc. Palo Alto, Calif.) for 3 minutes to pellet debris.Tubes were washed with an additional 150 ml of breaking buffer,supernatants collected in a 1.5 ml microfuge tube, and respun for 5minutes in an Eppendorf microfuge (Brinkinan). ELISA assays were begunthe same day.

5 ml of extract was diluted into 50 ml of 1% BSA in phosphate-bufferedsaline solution (PBS), aliquoted onto a 96 well Immulon 2 microtiterplate (Dynatech Laboratories, Inc.), and incubated overnight at 4° C.Extracts were removed and the plate blocked with 5% powdered milk/PBS.All subsequent wash steps were performed with 1% BSA/PBS. The plate wasincubated at room temperature with primary antibody for 1 hour. Theprimary antibodies, monoclonal antibodies CRPV.5A and H11.F1, wereobtained as ascites stock from Dr. Neil Christensen. They areVLP-dependent and type specific antibodies which recognize CRPV andHPV11 VLPs respectively (Neil Christiansen, personal communication).They were diluted 10⁵ -fold in 1% BSA/PBS before use. After washing in1% BSA/PBS, plates were incubated for 1 hour with secondary antibody,peroxidase labeled Goat anti-Mouse IgG (g) (Kirkegaard & PerryLaboratories, Inc.) and used at 10³ dilution in 1% BSA in PBS. After afinal washing, an alkaline phosphatase assay was performed andabsorbance read at 405 nm.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 6    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 27 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -      (v) FRAGMENT TYPE: internal    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    #Gly Tyr Gly Gly Asn Proal Glu Asn Ser Gly    #                 15    #Asply Gln Asp Asn Arg Val Asn Val Gly Met    #             25    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 27 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -      (v) FRAGMENT TYPE: internal    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    #Ala Tyr Ala Ala Asn Alahr Glu Asn Ala Ser    #                 15    #Asply Val Asp Asn Arg Glu Cys Ile Ser Met    #             25    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 26 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -      (v) FRAGMENT TYPE: internal    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    #Ser Ala Tyr Ala Ala Asnsp Thr Glu Asn Ala    #                 15    -  Ala Gly Val Asp Asn Arg Glu Cys Ile Ser    #             25    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 26 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -      (v) FRAGMENT TYPE: internal    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    #Gly Gly Tyr Ala Ala Asnsp Thr Glu Asn Ala    #                 15    -  Ala Gly Gln Asp Asn Arg Val Cys Ile Ser    #             25    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 26 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -      (v) FRAGMENT TYPE: internal    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    #Gly Gly Tyr Gly Gly Asnsp Thr Glu Asn Ala    #                 15    -  Pro Gly Gln Asp Asn Arg Val Cys Ile Ser    #             25    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 26 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -      (v) FRAGMENT TYPE: internal    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    #Gly Gly Tyr Gly Gly Asnsp Val Glu Asn Ser    #                 15    -  Pro Gly Gln Asp Asn Arg Val Cys Ile Ser    #             25    __________________________________________________________________________

What is claimed:
 1. Synthetic HPV16 virus-like particles selected fromthe group HPV16:8, HPV16:8L126Y, HPV16:8S133G, HPV16:8A134G,HPV16:8A136G, HPV16:8A137G, HPV16:8A139P, HPV16:8V141Q and HPV16:8E145V.